1. Field
The present invention relates to compositions and methods for treating osteoporosis and other bone degenerative diseases in subjects in need thereof, such as post-menopausal women.
2. Introduction
Bone is generally believed to be a tissue in a steady state, in which bone formation by osteoblasts and bone resorption by osteoclasts occur continuously. Osteoporosis is a disease that frequently occurs in menopausal and post-menopausal women, and is characterized by an imbalance of activity of osteoclasts and osteoblasts.
Recent evidence indicates that interleukin-17 (IL-17), which is produced primarily by the Th17 subset of CD4+ T cells but can also be produced by a variety of other cell types, is a key cytokine promoting osteoclast formation. IL-17 is an established inducer of receptor activator of NF-κB ligand (RANKL) expression in target cells such as osteoclasts (Gallen, S. L., Arthritis Res. & Therapy 6: 240-247, 2004; Udagawa, N., et al., Arthritis Res. 4:281-289, 2002).
In one recent study, ovariectomized mice genetically deficient for IL-17 Receptor (IL-17 RA−/− mice) were reported as exhibiting enhanced bone loss (Goswami, J., et al., Eur. J. Immunol. 39: 1-9, 2009). The authors of this report proposed that signaling via the IL-17 receptor simultaneously promotes osteoclastogenesis and inhibits adipogenesis. They concluded that IL-17RA signaling protects against estrogen deficiency-induced bone loss. These conclusions imply that promotion of IL-17RA signaling could be therapeutic for osteoporosis. However, their measurements used Dual energy X-ray absorptiometry (DEXA), which is not a precise measurement of bone volume or density in these mice because DEXA is influenced by fat distribution (Formica, C., et al., J. Bone Miner. Res. 10, 1504-1511, 1995; Hangartner, T. N., et al., Bone Miner. 9, 71-81, 1990; Tothill, P., et al., J. Bone Miner. Res. 12, 1908-1921, 1997; Tothill, P., et al., Br. J. Radiol. 72, 661-669, 1999; Wren, T. A., et al., J. Clin. Endocrinol. Metab. 92, 938-941, 2007).
Halofuginone, 7-Bromo-6-chloro-3-[3-[(2S,3R)-3-hydroxy-2-piperidinyl]-2-oxopropyl]-4-quinazolinone (including salts thereof), is a febrifugine analogoue and an established anti-coccidial agent approved by the USDA for the treatment of parasites in poultry and beef. Halofuginone inhibits IL-17 production and TH17 cell differentiation by activating the amino acid starvation response (Sundrud, M. S., et al., Science 324: 1334-1338, 2009). Halofuginone has use as an inhibitor of angiogenesis (US Patent Application Publication 20100029615 of Munchhof et al.). It has also been cited as an anti-sclerodermal agent (U.S. Pat. No. 7,638,480 to Power et al.) and as an inhibitor of collagen synthesis (Granot et al., Biochimica Biophys. Acta 1156: 107-112, 1993). It is also cited as an anti-cancer agent (U.S. Pat. No. 7,713,994 Tsou et al.) US Patent Application Publication 20090123389 of Whitman et al. discloses use of Halofuginone for treating cellulite.
The anti-sclerosis properties of Halofuginone are thought to arise from its ability to inhibit production of type I collagen by fibroblasts, which is the main constituent of fibrous tissue. Halofuginone inhibits collagen α1(I) mRNA and protein levels in a variety of cells, including mouse skin fibroblasts, avian, growth plate chondrocytes; a transformed rat embryo cell line, vascular smooth muscle cells, bovine aortic endothelial cells, and rat liver stellate cells (Bruck et al., Hepatology, 33(2), 379-386, 2001; Choi et al., Arch. Surg., 130(3), 257-261, 1995; Granot et al., Biochim Biophys Acta, 1156(2), 107-112, 1993). Collagen type II or III were not inhibited in these studies (Choi et al., Arch. Surg., 130(3), 257-261, 1995; Granot et al., Biochim Biophys Acta, 1156(2), 107-112, 1993). Halofuginone is shown to inhibit fibrosis accumulation in rat urethral stricture formation (Nagler et al., J. Urol., 164(5), 1776-1780, 2000), thioacetamide- and dimethylnitrosamine-induced rat cirrhosis (Bruck et al., Hepatology, 33(2), 379-386, 2001; Pines et al., J. Hepatol., 27(2), 391-398 1997), rat pulmonary fibrosis after bleomycin treatment (Nagler et al., Am. J. Respir. Crit. Care Med., 154(4 Pt 1), 1082-1086, 1996), and tight skin (Tsk)+ and cGvHD-afflicted mice (Levi-Schaffer et al., J. Invest. Dermatol., 106(1), 84-88, 1996; McGaha et al., J. Invest. Dermatol., 118(3), 461-470, 2002; Pines et al., Biochem. Pharmacol., 62(9), 1221-1227, 2001). The drug was effective whether given orally, locally, or intraperitoneally. The mechanism by which Halofuginone decreases collagen type I is unclear, but seems to require new protein synthesis, since cycloheximide or actinomycin D blocks the suppressive effect of Halofuginone on collagen α1(I) mRNA expression (Halevy et al., Biochem. Pharmacol., 52(7), 1057-1063, 1996). Its antifibrotic effects may be due to inhibition of TGFβ1 signaling (McGaha et al., J. Invest. Dermatol., 118(3), 461-470, 2002), but at concentrations that inhibit IL-17 production, no TGFβ inhibition is seen (Sundrud et al., Science, 324(5932), 1334-1338, 2009). Rather, Halofuginone induces the amino acid starvation response, which through unknown mechanisms prevents IL-17 production and Th17 development, a process that cannot be rescued by forced RORγt expression (Sundrud et al., Science, 324(5932), 1334-1338, 2009). In most animal models of fibrosis, regardless of the tissue, Halofuginone had a minimal effect on collagen content in the control, nonfibrotic animals, whereas it exhibited a profound inhibitory effect in the fibrotic organs. In culture, Halofuginone was effective in reducing collagen synthesis by fibroblasts after they had been stimulated with a profibrotic agents, but had a very small effect on collagen synthesis in control cells (McGaha et al., J. Invest. Dermatol., 118(3), 461-470, 2002). Even in animal models of pre-existing fibrosis, Halofuginone treatment can reduce fibrotic levels to normal levels (Nagler et al., Ann. Surg., 227(4), 575-582, 1998).
Given the promise of Halofuginone in treatment of systemic sclerotic conditions, Phase I-III studies have been performed using the drug topically, as well as a Phase I study for oral administration. The oral study was double-blind and involved 26 healthy, male volunteers receiving between 0.07 to 2.5 mg/d with food. Single, oral doses of 0.07 and 0.5 mg Halofuginone were found to be safe and well tolerated, with no clinically significant adverse events. At 1.5 to 2.5 mg, Halofuginone was moderately tolerated, with incidence of nausea and vomiting associated with dose, escalation. A daily dose of 1.5 mg Halofuginone was designated as the maximal tolerated dose. A later study found that dividing the dose into several daily portions allowed greater intake without increasing gastrointestinal adverse events (Pines et al., Biol. Blood Marrow Transplant, 9(7), 417-425, 2003).
Since earlier studies implicate IL-17 in the pathogenesis of osteoporosis, the inventors tested whether Halofuginone administration post-ovariectomy has an effect on IL-17 production and bone mass, and find Halofuginone to be a novel potential therapeutic for treatment of osteoporosis.
There are several structural analogues of Halofuginone that have been identified and/or synthesized, such as febrifugine, isofebrifugine, Df-1 and Df-2 (Takaya, Y. et al., J. Med. Chem 42: 3163-3166, 1999; Kikuchi, H., et al. J. Med. Chem. 45, 2563-2570, 2002). These molecules have use as anti-malarial agents (Takaya, Y. et al., J. Med. Chem 42: 3163-3166, 1999; McLaughlin, N. P., et al., J. Org. Chem. 2010 75: 518-521, 2010; U.S. Pat. No. 4,632,926 to Giarda et al.; U.S. Pat. No. 3,320,124 to Waletsky et al., U.S. Pat. No. 2,694,711 to Baker et al.)
Halofuginone and its analogs comprise a quinazolinone. Quinazolinones such as Halofuginone and its analogues have the general formula
wherein R1 is a hydrogen, a halogen, a nitro, a benzo, a lower alkyl, a phenyl, or a lower alkoxy and can be located at one or more of the 6, 7 or 8 positions on the quinazolinone nucleus; R2 is a hydroxyl, an acetoxy, or a lower alkoxy; and R3 is a hydrogen or a lower alkoxycarbonyl; lower alkyl and lower alkoxy radicals can have from 1 to 6 carbons (U.S. Pat. No. 3,320,124).
Halofuginone and its analogues have also been described as
wherein R′═H, halogen, nitro, benzo, alkyl, phenyl or alkoxy; R″=hydroxy, acetoxy or alkoxy; and R′″═H or alkenoxycarbonyl (U.S. Pat. No. 4,632,926). Additional examples of quinazolinone analogues of Halofuginone which are active against coccidiosis include
wherein R is an alkyl C1-C4, an alkoxy C1-C4, an alkylthio or a halogen; n is zero, one or two; R1 is a hydrogen atom or an alkyl C1-C4; R2 is a hydrogen atom, an alkyl C1-C8, a cycloalkyl C3-C6 or a phenyl optionally substituted by one or more alkyl C1-C4 or halogen atoms (U.S. Pat. No. 4,632,926).
Kikuchi, H., et al., J. Med. Chem. 45, 2563-2570, 2002 have described several analogues of Halofuginone, including analogues of febrifugine, such as the following compounds:

These workers indicated that the 3″-keto analogue of febrifugine (7) was found to exhibit potential antimalarial activity with high selectivity against P. falciparum in vitro. The in vitro activities of the reduction product (8, EC50=2.0×10−8 M) of febrifugine at C-2′ and its cyclic analogues 9 and 10 (EC50=3.7×10−9 and 8.6×10−9 M, respectively) were found to be strongly active and selective. Additionally, the Dess-Martin oxidation product of 3 was found to be strongly active with high selectivity against P. falciparum. The authors concluded that a structure-activity relationship study (SAR) demonstrated that the 4-quinazolinone ring and the presence of a 1″-amino group and C-2′, C-3″ O-functionalities are crucial in the anti-malarial activity of febrifugine. Anti-malarial activities of febrifugine, isofebrifugine, Df-1, Df-2 and various derivatives thereof are listed in table 1 and table 2.
TABLE 1Antimalarial Activities of Febrifugine (1)and Isofebrifugine (2) Derivatives in VitroEC50 (M)antimalarialcompoundactivityacytotoxicitybselectivityc1 7.0 × 10−101.7 × 10−724323.4 × 10−91.8 × 10−75359.1 × 10−7>2.9 × 10−5 >3264.8 × 10−6>1.7 × 10−5 >3.572.0 × 10−81.0 × 10−550082.0 × 10−81.5 × 10−575093.7 × 10−93.8 × 10−61027108.6 × 10−92.5 × 10−6291118.4 × 10−7>2.5 × 10−5 >30126.0 × 10−7>1.9 × 10−5 >32134.0 × 10−87.0 × 10−6175145.0 × 10−7>1.6 × 10−5 >32152.1 × 10−6>6.3 × 10−6 >3chloroquine1.8 × 10−83.2 × 10−51778artemisinin1.0 × 10−81.0 × 10−51000aAgainst P. falciparum FCR-3.bAgainst FM3A mouse mammary cells.cCytotoxicity/antimalarial activity.
TABLE 2Antimalarial Activities of Df-1 (3)and Df-2 (4) Derivatives in VitroEC50 (M)antimalarialcompoundactivityacytotoxicitybselectivityc31.6 × 10−9 3.8 × 10−723842.8 × 10−9 2.4 × 10−6857291.9 × 10−9 5.9 × 10−6>3105304.0 × 10−7 2.8 × 10−670313.0 × 10−7 8.5 × 10−5283323.6 × 10−9 1.3 × 10−6361358.3 × 10−7>2.2 × 10−5>27364.8 × 10−6>3.2 × 10−5>7371.3 × 10−6>6.6 × 10−5>51384.2 × 10−7>1.6 × 10−5>38396.0 × 10−7>1.7 × 10−5>28401.0 × 10−7>2.9 × 10−5>290418.0 × 10−7>2.4 × 10−5>30423.4 × 10−6>1.0 × 10−4>28434.0 × 10−7>1.1 × 10−5>28447.0 × 10−6>2.1 × 10−5>3451.9 × 10−8 7.0 × 10−6368aAgainst P. falciparum FCR-3.bAgainst FM3A mouse mammary cells.cCytotoxicity/antimalarial activity.
U.S. Pat. No. RE39,574 E to Pines et al. describes using quinazoline compounds such as Halofuginone for attenuating neovascularization in the treatment of certain malignancies.
There is an ongoing need for drugs and therapies which can be used to treat degenerative bone diseases such as osteoporosis.